Since isolation of vascular endothelial growth factor (VEGF), the complicated network of extracellular signal transduction in angiopoiesis has been drastically elucidated. Research into mice which has a targeted mutation for the tyrosine kinase, for example, this ligand, Flk-1/VEGFR2, and Flt-1/VEGFR1, etc. was a first key point related the first step of angiopoiesis and vascularization.
VEGF receptor-2 (VEGFR-2) is necessary in endothelial formation and a targeted mutation for this gene resulted in a shortage of blood cells or organized blood vessels and death in the embryo at 9.5 days post-coitum. However, according to analysis of early marker by RT-PCR revealing early hemopoiesis and the report that endothelial precursory cell was actually formed in absence of VEGFR-2, it is suggested that VEGFR-2 is required for the growth later, but not absolutely essential for hematoblast formation.
VEGF receptor-1 (VEGFR-1) knock-out mice were also dead in the embryo at 9.5 days post-coitum, but differentiation of their endothelial cells was not influenced directly. Instead, it is speculated that movement of the mesenchyme hematoblasts may be stagnant generally and that their overcrowded endothelial precursory cells may result in destroying vascular system severely.
VEGF knock-out mice were also dead in the embryo at 9.5 days post-coitum conspicuously, and differentiation of their endothelial cells were not defective. That is, substantially, it was the same to VEGF-1 knock-out mice, but the phenotype was slighter than that of VEGF-1 knock-out mice. The conspicuous characteristic related to a targeted mutation within this gene was that gene deletion of the heterozygote was lethal to the mouse embryo at 11.5 days post-coitum. This revealed that development in the embryo may depend strictly on the quantity of VEGF expression.
Other tyrosine kinase receptors, such as Tie-2 and its ligand, are specifically expressed in vascular endothelial cells, contributing to the step of vascularization in the late stage of angiopoiesis. Though destruction of these genes, which does not have any influence on angiopoiesis, affects vessel remodeling to cause death in the embryo at 10.5 days post-coitum. They play some roles in interaction between endothelial cells and their surrounding vascular smooth muscle cells (VSMC) or mesenchyme cells. In addition to the system specific to vascular, PDGF-BB, TGF-β and their receptors also play similar roles in interaction between endothelial cells and their surrounding cells. Investigations into the relationship between vascular system and neuropilin-1 (NP-1) which was cloned as an antigen recognized by the monoclonal antibody recognizing the first developed neuron led this field to a new phase.
Then, identification as a receptor of semaphorin 3A (sema3A) has come to attract attention in relation to axon formation, and search for other VEGF receptors led to discovery of NP-1 which is also a co-receptor for VEGFR-2. NP-1 enhances not only the binding to one isoform of VEGF-2, but also chemotaxis and probable mitosis of endothelial cells. Before this identification, it was revealed that chimerical mice over-expressing NP-1 presented the phenotype of hypertrophic vascular formation. NP-1 knock-out mice presented the phenotype of low angiogenesis in the central nervous system and large vascular variations. On the other hand, it has not been reported whether sema3A knock-out mice presented the abnormal vessels, while it is interesting that these mice presented the phenotype of thin myocardium, which was also observed in mice over-expressing NP-1.
Another example is the Eph/Ephrin system, which was first studied extensively in the neurological field. Later, this system was re-identified as an only surface marker discriminating arteries and veins. They are expressed in mesenchyme cells which surround the vessels and contact with endothelial cells. Speculating that there are many extracellular signal transduction molecules in vessels besides the above molecules, the inventors have suggested that proving this speculation would contribute to more understanding of this complex system.